The present invention relates, in general, to novel methods for measuring cell movement and to methods for assessing the impact of a variety of factors on the ability of cells to move. In particular, the present invention relates to systems and methods for measuring cell movement and the effect of various chemical species on that phenomenon by monitoring one or more electrical parameters of sensing electrodes that are sensitive to the interaction of cells with the electrode surface.
Cells move from place to place in multicellular organisms for a variety of reasons. For example, cell movement occurs during organogenesis, movement is essential to inflammatory immune responses, and movement of neoplastic cells permits metastasis to secondary sites. This movement can arise from the intrinsic characteristics of the cell, or it can be initiated, enhanced or otherwise affected by the presence of external chemical stimuli. Stimuli can be divided into two classes: those that stimulate cell movement without a specific directional aspect (chemokinesis), and those stimuli that enhance directional cell movement according to the location of external cues (chemotaxis).
Cell movement is a critical component of both normal immune function and the dysfunctional immune responses associated with certain disease conditions such as asthma, chronic inflammation and autoimmune disease. Often this movement is characterized as chemotactic, and is initiated in response to the presence of one or more of a set of chemoattractants. These chemoattractant species (e.g., chemokines and activated components of the complement cascade) can be produced by the body in response to a variety of stimuli. Additionally, cells of the immune system are capable of rapid and vigorous responses to stimuli provided by infectious microorganisms (e.g., f-met-leu-phe and other formulated peptides that are products of bacterial protein degradation). In the context of an infection or other inflammation, these signals cause the influx of cells (notably macrophages and neutrophils) at the site of inflammation. The common mechanism of action for these signals is to engage specific receptors on the surface of the cell. Following ligand/receptor engagement, one or more signal transduction cascades are initiated, and the cell responds by specific activation of genes and the movement of the cell along the gradient. Still unknown is the means by which these cells sense the gradient, and the actual mechanisms by which they move through the cellular environment to arrive at the source of the chemoattractant.
From a practical standpoint, studies that identify new chemokines and other attractants, that characterize the signal transduction cascade and the differentiation of the responding cell, as well as studies that characterize the environment in which movement occurs, will each provide potential avenues of therapeutic manipulation. Assays that measure cellular movement in response to a chemotactic gradient offer the ability to assess individual elements along the length of the path from initiation of the response to the cellular accomplishment of the movement.
Quantitative and qualitative measurement of cell movement can be important to the characterization of biological responses, such as those mentioned above, as well as to many others. The rational design of therapeutic strategies for clinical intervention in these systems can theoretically depend upon manipulation of cellular motility: increasing it when a more robust response is desired, and diminishing the influx of cells to reduce their contributions to the response. For example, pharmacological manipulations of cell accumulation in the airways has been found to be an effective treatment for some forms of asthma, and interference with cellular movement through the vascular epithelia can diminish some the inflammatory damage associated with ischemia/reperfusion injury. Vigorous research efforts are currently underway in many biotechnology and pharmaceutical laboratories to discover novel therapeutics with the capacity to affect cell movement. For example, it is understood in the art that a potential therapeutic approach is to use inhibitors of signal transduction to manipulate chemotactic responsiveness, and many investigations are currently under way to assess the viability of such an approach in the treatment of a number of disease conditions. Essential to these investigations is the capacity to make qualitative and quantitative observations of cell movement in response to chemotactic stimuli, as well as mediation of that response by inhibitors or enhancers of chemotactic response.
In the prior art, measurement of cell movement directed by chemotactic agents has been accomplished in several ways. A xe2x80x9csmall-populationxe2x80x9d assay can optically measure the movement of cells in an initial localized deposit of these cells in a chemotactic gradient that exists in proximity to the cells. Variations of the Boyden chamber assay (Boyden, S., Journal of Experimental Medicine 115: 453 (1953)) are currently the most commonly used. In these assays, the cells are placed on a microporous membrane over a source of chemotactic agent. As the cells detect the higher concentrations of chemotactic agent that diffuse from the source, they migrate through the membrane to its underside. Migrating cells are usually statically detected by manual and optically aided methods on the reverse side of the membrane after staining. The quantity of responding cells is usually determined as an endpoint assay at a predetermined time-point. Thus, assays of this sort are usually capable of a semi-quantitative measure of the number of cells from an initial cell population that travel across a membrane in response to a perceived gradient of a chemoattractive agent. An advantage of this type of technique is the ability to perform many simultaneous assays, as multi-well plates in a two-dimensional array may be effectively utilized. However, a major limitation of a Boyden-type assay is that the chemical gradient sensed by the cells is very steep and dissipates rapidly. Essentially, there is a high concentration of chemoattractant on one side of the separating membrane and none on the other. In addition, it is also difficult to visualize the movement of cells through the membrane in this chemotactic environment. Finally, quantitation of the number of cells to move in response to the chemotactic stimulus is limited by traditional cell-counting methodologies and other errors inherent in the system such as the loss of migrated cells from the underside of the membrane where counting occurs.
Another technique used to measure chemotaxis is to track cell movement by video microscopy in a Zigmond or Dunn chamber. In these assays, the movement of cells is recorded as they respond to an aqueous gradient of chemoattractant formed between two closely spaced glass surfaces. This assay suffers from serous drawbacks in that it is more difficult to set up, only a small number of cells can be analyzed at one time, and the assay cannot be easily multiplexed.
The under-agarose chemotactic assay (Nelson, R. D., et al. Journal of Immunology 115: 1650-1656 (1975)) provides a different approach from that offered in other, Boyden-type assays. In the under-agarose assay, a planar layer of agarose gel is cast in a cell culture dish. Multiple wells are cut in the agarose layer with a device such as a stainless steel punch. In a typical assay, multiple sets of three wells are punched in a linear array. In the middle well of the three-well set, a portion of cell suspension is added to the well. In one of the adjoining wells, a solution of a chemotactic agent is added. In the third well, a suitable control solution is added. The assembly is allowed to incubate at an appropriate temperature for a pre-determined period of time after which the cells are fixed with the agarose layer in place by the addition of suitable fixing agents such as absolute methanol. After fixation, the gel layer is removed and the plates stained. The migration patterns of the cells are observed optically and measurements taken of individual cells along paths toward the chemoattractant well and compared with the movement of cells toward the control well.
This type of assay provides a significantly different type of cell environment than that utilized in a Boyden-type assay. First, the cells under investigation move while surrounded by the underlying substrate (glass culture dish) and the overlying agarose layer. Second, the chemotactic gradient is stabilized by the agarose allowing the gradient to be established over a larger volume, and for a longer period of time. As indicated above, the under-agarose assay measures the distance cells move in a specified period of time as an indication of a chemotactic response. This assay has the advantage that a single endpoint need not be evaluated since the cells gradually spread away from the starting well. The disadvantage is that in running many parallel assays, each would have to be evaluated microscopically at many time points to get an estimate of the extent of movement in each assay. Furthermore, the nature of the measurements obtained with this assay render it very difficult to quantify the rate at which cells move in response to chemotactic stimuli.
In the interest of obtaining information on a totally different type of cell motion, Giaever and Keese have developed an electrochemical-based system for assessing cell motility, as disclosed in U.S. Pat. No. 5,187,096, the disclosure of which is hereby incorporated in its entirety. A commercialization of this system, known as the Electric Cell Impedance Sensing (xe2x80x9cECISxe2x80x9d) system has been developed and is sold by Applied Biophysics, Inc. (Troy, N.Y.).
In the ECIS assay system, two electrodes are lithographed onto the surface of a lexan slide and positioned within a chamber that holds aqueous media. Cells in this media can attach to a sensing electrode and to the surrounding surface of the slide. A 1 volt a.c. current passes through the culture media that functions as an electrolyte, and a lock-in amplifier measures current flow through this circuit. This measurement provides data on the initial resistance of the system and, more importantly, any changes to current flow on the electrode that occur over time. Due to the relatively small size of the electrode, resistance at the sensing electrode predominates in the system. Any activity that affects the adherence of cells to the electrodes will alter the measured electrical resistance in the system. For example, increasing the tightness of association of cells with the surface of the electrode by coating it with extracellular matrix proteins increases the resistance of the electrical circuit. Lipopolysaccharide (LPS) activates macrophages to spread and cover a larger amount of the target electrode and thus also increase the resistance measured at the target electrode. In contrast, toxicants that damage cells will act to reduce the resistance of the circuit. The degree of or changes in cell motility will also be reflected by changes in the measured electrode resistance as the extent of interaction between the cells and the electrode surface changes.
FIG. 1 illustrates a typical prior art ECIS configuration with a side view (not to scale) of cells 54 sitting on the sensing electrode 10 in a culture well 50. The electrodes comprise gold electrodes fabricated on plastic substrata 58. Culture media 55 is used as the electrolyte. In a typical ECIS application, a constant AC current of 1 microampere at 4 kHz is maintained between the sensing electrode 10 and a large counter electrode 40, while the voltage is monitored with a lock-in amplifier 52. Voltage and phase data are stored and processed with a microprocessor 60. Normally, these data are converted to resistance or capacitance by treating the cell-electrode system as a series RC circuit. The same microprocessor controls the output of the amplifier and switches the measurement to different sensing electrodes in the course of an experiment with a multi-cell array.
In an ECIS system, the relative sizes of the sensing and counter electrodes can be significant. With larger sensing electrodes, cell-related resistance signals become difficult to detect. This is a consequence of bulk solution resistances that tend to swamp out the contribution to total resistance from the sensing electrode. When electrodes have a surface area of approximately 10xe2x88x923 cm2 or less, the impedance of the electrode-electrolyte interface at 4 kHz predominates, and in this situation, changes in resistance due to interaction of the cells with the electrode surface are clearly revealed.
In a typical assay, cells seeded into an ECIS well settle to the bottom of the well, attach to the surface of the sensing electrode 10 that is fabricated on the bottom surface of the well, and individual cells spread radially. The number of cells on the well, the intimacy of contact, the degree of spreading, and the activity (motility) of the cells all contribute to the level of resistance imparted by the cells to the circuit. A single electrode can be monitored as often as four times per second with currently available hardware in the commercial embodiment of the ECIS system. The intimacy of cell contact with the electrode can be modified by pre-incubation of the electrode with different extracellular matrix proteins and this can result in different levels of resistance imparted by the cells to the system. Moreover, the intimacy of contact can be modified by exposing the cells to agents that alter the viability, signal transduction, or membrane integrity of the cell.
As disclosed in U.S. Pat. No. 5,187,096, cited above, the ECIS system is directed toward investigations of cellular phenomena that are only remotely implicated in the type of cell movement associated with chemotactic or chemokinetic behavior. As such, its utility, although specialized, does not extend in its conventional applications to investigations into the mechanism of translational cell movement, or the influence of chemical agents on that motion.
Consequently, there exists a need in the art for an assay system directed toward translational cell movement that is capable of rapid, automated and multiplexed analysis of cell movement and factors capable of affecting such movement. In a unique combination of the traditional under-agarose cell assay with the specific capabilities of an ECIS system, the present inventors have developed a system and methods for investigation of phenomena associated with cell movement that possesses these desirable characteristics, and addresses the majority of the shortcomings associated with prior art techniques. Specific embodiments of these systems and methods are detailed below.
In a first embodiment, the present invention provides a system for monitoring the effect of extracellular chemical stimuli on the translational motion of cells, the system comprising: (a) an array of one or more cell containment volumes; (b) an array of one or more chemical agent volumes interspersed among the array of one or more cell containment volumes; (c) one or more substantially planar sensing electrodes distributed within the arrays of cell containment volumes and chemical agent volumes so that at least one of the sensing electrodes is between one cell containment volume and one chemical agent volume, wherein the one or more sensing electrodes is operatively coupled to a sensing device capable of measuring an electrical parameter of the sensing electrode; (d) at least one counter electrode in electrical connection with the one or more sensing electrodes; and (e) a biocompatible chemical gradient stabilizing medium in simultaneous diffusional contact with the arrays of cell containment volumes and chemical agent volumes. In this embodiment of the present invention, the at least one counter electrode and the one or more sensing electrodes are connected in series.
In addition, the present invention contemplates a system wherein the at least one counter electrode and the one or more sensing electrodes are connected in parallel. Alternatively, the at least one counter electrode and the one or more sensing electrodes are connected in series. Also, the system of the invention can further comprise a reference electrode in electrical connection to the at least one counter electrode and the one or more sensing electrodes. The system of the invention also contemplates that the measured electrical parameter of the sensing electrode is impedance, or resistance, or capacitance.
As exemplified in this embodiment, the system of the invention contemplates a chemical gradient stabilizing medium that is in a planar geometry overlying the arrays of cell containment volumes and chemical agent volumes. Preferably, the chemical gradient stabilizing medium is an agarose gel. Furthermore, preferentially, the geometry of the sensing electrode is substantially circular. Alternatively, the geometry of the sensing electrode can be substantially rectangular. It is also possible that the geometry of the sensing electrode is semi-circular. Preferably, the surface area of each of the one or more sensing electrodes is from about 0.5xc3x9710xe2x88x922 mm2 to about 10xc3x9710xe2x88x922 mm2.
In a particularly preferred embodiment, the system of the present invention comprises a sensing device that is operatively coupled to a microprocessor. This microprocessor can be connected to an output display device capable of displaying the electrical parameter values measured at the one or more sensing electrodes. Preferably, the output display device is a cathode ray tube (CRT), or alternatively, a hard copy device such as plotter or printer. More preferably, the microprocessor is under the control of a software program executable on the microprocessor.
In yet another embodiment, the present invention provides a method for monitoring the translational motion of cells in response to extracellular chemical stimuli, the method comprising the steps of (a) placing a population of one or more cells in a biocompatible medium into a cell containment volume; (b) placing a chemical agent in a biocompatible medium into a chemical agent volume in diffusional contact with a biocompatible chemical gradient stabilizing medium; and (c) monitoring changes in an electrical parameter of one or more substantially planar sensing electrodes interposed between the cell containment volume and the chemical agent volume and in electrical connection with a counter electrode, wherein the changes in electrical parameter of the one or more sensing electrodes arise substantially from contact of one or more cells from the cell population with a surface of one or more of the sensing electrodes, and wherein the one or more cells have diffused to the surface of one or more of the sensing electrodes from the cell containment volume under the influence of a chemical gradient of the chemical agent in the chemical gradient stabilizing medium.
According to the present embodiment of the claimed invention, the measured electrical parameter is impedance. Alternatively, the measured electrical parameter is resistance or capacitance. In another aspect of this embodiment, the translational movement of the one or more cells is directionally focused. Alternatively, the translational movement of the one or more cells is not directionally focused. In yet another aspect of this embodiment, there is additionally interposed between the cell containment volume and the one or more sensing electrodes one or more barriers to translational motion of the cells. The present invention contemplates that the barrier is physical in nature. Alternatively, the barrier may be chemical in nature. In an alternative configuration of this embodiment of the claimed invention, the sensing electrode and the counter electrode are in electrical connection with a reference electrode.
In the practice of the present invention, the one or more cells are exposed to two or more independent chemical gradients from different chemical agents. In this aspect, the independent chemical gradients are physically overlapping. Alternatively, the independent chemical gradients are not physically overlapping.
According to this embodiment of the present invention, the cells of the cell population are selected from the group consisting of D. discoideum, bone marrow cells from BALB/c mice, M1 cells, U937 cells, and other motile eukaryotic cells from both tissue culture and from living animals.
In addition, in the practice of the claimed invention, the chemical agent may be selected from the group consisting of folic acid, guinea pig serum, activated complement, bacterial peptides, and mammalian chemokines.
The present embodiment also contemplates that the chemical agent volume is the biocompatible chemical gradient stabilizing medium.
In still another embodiment, the claimed invention provides a method for determining the impact of a test substance on the ability of a chemical agent to affect the translational movement of cells, the method comprising the steps of (a) placing a population of one or more cells in a biocompatible medium into a cell containment volume; (b) placing a chemical agent in a biocompatible medium into a chemical agent volume in diffusional contact with a biocompatible chemical gradient stabilizing medium; (c) exposing one or more cells of the population to a test substance; (d) monitoring one or more electrical parameters measured on a substantially planar sensing electrode positioned between the cell containment volume and the chemical agent volume, wherein the changes in impedance on the sensing electrode arise substantially from contact of one or more cells from the cell population with a surface of the sensing electrode, and wherein the one or more cells have diffused to the surface of the sensing electrode from the cell containment volume under the influence of a chemical gradient of the chemical agent in the chemical gradient stabilizing medium between the cell containment volume and the chemical agent volume; and (e) comparing the one or more electrical parameters measured in step (d) with electrical parameter measurements taken for one or more cells from the population that have not been exposed to the test substance.
As practiced, the method of the present invention contemplates that the measured electrical parameter is impedance. Alternatively, the measured electrical parameter is resistance or capacitance.
In addition, the method of the claimed invention further contemplates exposing the cells to a second test substance and comparing the resulting measured electrical parameter readings with corresponding electrical measurements taken for one or more cells from the population that have been exposed to the first test substance but not the second test substance.
In one aspect of this embodiment of the claimed invention, the translational movement of the one or more cells is directionally focused. Alternatively, the translational movement of the one or more cells is not directionally focused. In yet another aspect, there is additionally interposed between the cell containment volume and the one or more sensing electrodes one or more barriers to translational motion of the cells. The one or more barriers may be physical in nature and/or chemical in nature.
In an alternative embodiment, the present invention provides a system for the non-optical imaging of translational cell movement comprising (a) one or more cell containment volumes; (b) one or more chemical agent volumes; (c) a plurality of sensing electrodes interposed between the cell containment volumes and the chemical agent volumes, wherein each of the plurality of sensing electrodes is operatively coupled to a sensing device capable of measuring an electrical parameter of the sensing electrode; (d) at least one counter electrode in electrical connection with the array of sensing electrodes; and (e) a biocompatible chemical gradient stabilizing medium in simultaneous diffusional contact with the cell containment volumes and the chemical agent volumes. Preferably, the plurality of sensing electrodes are arranged in an orderly, two-dimensional array. In this embodiment, the dimensions of individual sensing electrodes is of an order that is not much larger than the dimensions of a typical cell such that the electrode surface is large enough to hold only one cell at a time. As configured, this embodiment of the invention comprises an array of at least 100 sensing electrodes. Preferably, the array comprises at least 1000 sensing electrodes. More preferably, at least 2500 sensing electrodes.
In this embodiment the electrical parameter measured at the sensing electrode is impedance. Alternatively, the electrical parameter is resistance or capacitance. This embodiment of the invention also contemplates the further inclusion of a reference electrode in electrical connection to the at least one counter electrode and the array of sensing electrodes. In addition, the present invention provides that the chemical gradient stabilizing medium is in a planar geometry overlying the arrays of cell containment volumes and chemical agent volumes. Preferably, the chemical gradient stabilizing medium is an agarose gel.
In a particularly preferred embodiment, the system of the present invention comprises a sensing device that is operatively coupled to a microprocessor. This microprocessor can be connected to an output display device capable of displaying the electrical parameter values measured at the one or more sensing electrodes. Preferably, the output display device is a cathode ray tube (CRT), or alternatively, a hard copy device such as plotter or printer. More preferably, the microprocessor is under the control of a software program executable on the microprocessor.